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Maywood I is a dysfunctional plasminogen. It is described in a patient (W.Y.) with a reduced plasma functional activity and with a low normal antigen level. Plasminogen was isolated from the patients plasma by affinity chromatography with L-lysine-substituted Sepharose. The protein yield was 86 mg/l, which was 88% of the plasma Plg antigen level; the specific activity was 24.4 IU/mg protein compared to 28.5 IU/mg protein for the native molecule. The protein was the Glu-form determined by SDS-PAGE and by isoelectric focusing. Six major isoelectric forms were found with isoelectric points between pH's 6.40 and 5.45. Titration of the equimolar plasminogen.streptokinase complex with p-nitrophenyl-p-guanidinobenzoate gave 85% active-sites indicating a homogenous population of molecules; therefore, the propositus is a homozygote. Four different plasminogen activators: a) streptokinase, b) urokinase c) the plasmin-derived light (B) chain-streptokinase complex, and d) tissue plasminogen activator (with soluble fibrin/CNBr-fibrinogen fragments) generated little plasmin from the variant plasminogen (4.5 to 45 nM), 5% or less than that generated from normal plasminogen. At 45 nM plasminogen, the molar ratio of plasminogen:activator was 3.0 for streptokinase, 3.9 for urokinase, 7.1 for the light (B) chain-streptokinase complex, and 155 for tissue plasminogen activator. In the equimolar variant plasminogen.streptokinase complex, the active-site was slowly developed, to a maximum of 85% in 40 min; in the normal complex, 100% active-sites were developed in 15 min. The variant plasminogen forms two equimolar complexes with streptokinase (I and II), with different mobilities in PAGE, in about equal amounts.(ABSTRACT TRUNCATED AT 250 WORDS)

A new abnormal plasminogen, Frankfurt I, has been identified in the plasma of a 42 year-old male patient who has recurrent deep vein thrombosis. Clinical laboratory data showed normal hemostasis test results. Since plasma plasmin generation rates gave low values, the fibrinolytic system was analyzed for a possible fibrinolytic system defect. Functional and antigen plasminogen concentrations both in the plasma and with the isolated, purified plasminogen showed that only 49% of the antigen concentration had potential functional active sites. Also, a reduced antigen concentration was found in both the propositus, and his mother (46% active sites). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified Frankfurt I plasminogen showed a normal native Glu-plasminogen band. Crossed-immunoelectrophoresis revealed a peak with normal size and shape, but displaced with respect to normal Glu-plasminogen toward the anode, i.e., was, as a whole, more negatively charged. Isoelectric focusing followed by zymography on a agarose-fibrin plate proved this observation, but did not indicate a separation of the normal from the abnormal plasminogen molecular species, also, fewer bands were found in the abnormal plasminogen isozyme pattern. Kinetic studies of Frankfurt I Glu-plasminogen and plasmin led to the conclusion that most of the functional abnormality is related to absence of active sites in half of the molecules. The plasmin generated was very unstable in the absence of stabilizing ligands and/or substrates. After reduction, the plasmin was completely converted to the typical two plasmin chains, A and B.

Oral administration of clinical-type high molecular weight urokinase (HMW-UK) in a single dose of 30,000-60,000 International Units (IU) in enteric-coated capsules, in a group of normal human subjects, induced a plasma fibrinolytic state suggesting transport of HMW-UK across the intestinal tract. Other groups of human subjects were given a single dose of 120,000 IU daily of pure HMW-UK for 1 d and 7 d together with a placebo dose, all of the ingredients except urokinase (UK), daily for 7 d. UK-type proteins were isolated from the plasma of blood samples drawn 6 h after administration of the final dose, by a sequential two-step affinity chromatography method first with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose and second with a specific rabbit anti-HMW-UK-IgG-Sepharose. The yield of UK-type proteins from the 7-d group, 0.79 mg/dl, was approximately twofold greater than that obtained from either the placebo or 1-d groups. The specific plasminogen activator activity of the 1-d and 7-d groups were similar, 508 and 537 IU/mg protein, respectively; negligible plasminogen activator activity could be detected in the placebo group. The kinetics of activation parameters of human Glu-plasminogen of the UK-type protein, isolated from the 1-d group, were similar to those obtained with urinary HMW-UK. The UK-type proteins isolated only from the 7-d group showed, in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a major protein band of molecular weight 53,000 in the same position as HMW-UK. In addition, two other major protein bands of approximately 140,000 and approximately 120,000 mol wt were found in the 7-d placebo-, and 1-d groups, and also in the 7-d group. The 53,000 mol wt protein, about 50% of the total protein in the 7-d group, was further purified by preparative SDS-polyacrylamide gel electrophoresis, and found to be a two-chain protein with a specific activity of 1,241 IU/mg protein. The protein showed common antigenic determinants with urinary HMW-UK. The oral administration of 120,000 IU HMW-UK to human subjects for 7 d stimulated the synthesis of a UK-type protein of 53,000 mol wt, probably the zymogen, from either the liver or vascular endothelium, which was released into the circulation, and converted into active UK by circulating plasmin. The induction of the fibrinolytic state produced the conversion of native circulating Glu-plasminogen into the degraded Lys-plasminogen form, which was found in large amounts in the plasma of the 7-d group. The new plasma components, e.g., the 53,000-mol wt UK-zymogen and Lys-plasminogen, could play an important role in clot resolution of the fibrin-thrombus in thromboembolic diseases. Oral administration of HMW-UK has been shown to be clinically effective in patients with cerebral thrombosis in a multicenter double blind study.

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.

A comparison was made of the esterase and activator activities of the various activated forms of human plasminogen and their streptokinase complexes with Nalpha-Cbz-L-lysine-p-nitrophenyl ester as the substrate. The steady state kinetic properties of Glu- and Lys-plasmins, and Glu- and Lys-plasminogen-streptokinase complexes were identical, while the Lys-plasmin-streptokinase complex showed a 2-fold increase in Km with the same kcat and a 3-fold increase in Ki for the competitive inhibitor leupeptin. Lys-plasminogen (zymogen with an active site) was prepared which incorporated 0.7 mol of [3H]idisopropyl phosphorofluoridate and 0.43 mol of p-nitrophenyl-p'-guanidinobenzoate/mol of protein. The Km for Lys-plasminogen was 3-fold higher than that of Lys-plasmin, and its maximum velocity 10-fold lower. The steady state kinetic parameters of a plasmin-derived light (B) chain (CmCys)3, and a derived equimolar light (B) chain-streptokinase complex (CmCys)3, isolated from human plasmin and equimolar plasmin-streptokinase, or plasminogen-streptokinase, complexes, respectively, were determined. When the light (B) chain-streptokinase complex is isolated from its parent complexes, there is a complete retention of the original parent's esterase activities, with respect to Km and kcat, and interaction with the competitive inhibitors benzamidine and leupeptin. The plasmin-derived light (B) chain does not retain its parent esterase activities. This chain has very similar kinetic properties to Lys-plasminogen except that streptokinase, in an equal molar amount, does not impart full esterase activity to the light (B) chain whereas the zymogen can be completely activated by streptokinase. The kcat of the plasmin-derived light (B) chain, and its streptokinase complex can be enhanced by 50 and 30%, respectively, in the presence of 10(-4) M leupeptin, a competitive inhibitor of plasmin, attesting to the increased structural flexibility within the active site of this enzyme species. Urokinase hydrolyzes Nalpha-Cbz-L-lysine p-nitrophenyl ester efficiently with a kcat/Km of one-third that of plasmin. The human plasminogen activator activities of various activated forms of human plasminogen and their equimolar streptokinase complexes were compared in a kinetic assay. The Lys-plasmin-streptokinase complex, and streptokinase were the least active of the activator species and were approximately equal in their activator activities. Glu- and Lys-plasminogen-streptokinase complexes had approximately 1.5 times the activity of streptokinase, whereas the equimolar light (B) chain-streptokinase complexes had approximately 2- to 3-times the activator activity of streptokinase. Since the esterase activity remained unchanged, this indicates a greater degree of specificity in the active site of the equimolar light (B) chain-streptokinase activator complex. Urokinase proved to be a poor activator species...